This application is the U.S. national stage application of International Application PCT/GB98/00616, filed Feb. 26, 1998, which international application was published on Sep. 3, 1998, as International Publication WO 98/38215 in the English language. The International Application claims priority of Great Britain Patent Application 9704174.3 filed Feb. 28, 1997.
This invention relates to an agent for alleviating the effects of muscular dystrophy and other muscle-wasting diseases (eg. motor neurone disease) in which there is leakage of enzymes such as creatine phosphokinase (CPK) from the muscle into the blood (hereinafter simply called xe2x80x9ca muscle-wasting diseasexe2x80x9d).
The present invention resides in the use of an agent which is an active xcex2-endorphin fragment or an analogue of such a fragment, in the manufacture of a medicament for alleviating the effects of muscle-wasting disease.
The present invention also resides in the use of an agent which is an active xcex2-endorphin fragment or an analogue of such a fragment, in the manufacture of a medicament for inhibiting or reducing leakage of an enzyme (preferably CPK) from a living muscle.
The present invention also resides in the use of an agent which is an active xcex2-endorphin fragment or an analogue of such a fragment, in the manufacture of a medicament for improving the contractile response of muscle.
xcex2-Endorphin is a 31 amino acid peptide which is processed in vivo by cleavage of basic amino acid residues to xcex2-endorphin1-7, and it has been assumed that cleavage of adjacent basic residues results in the production of the C-terminal dipeptide, xcex2-endorphin30-31.
The effect of these active xcex2-endorphin fragments is particularly surprising because it has previously been observed (see Smith, M. E. and Hughes, S., xe2x80x9cThe Effect of xcex2-Endorphin and xcex1-Melanotropin on Muscle Wasting in Micexe2x80x9d, Journal of the Neurological Sciences (1995),129 (suppl.), p127) that intravenous injection of xcex2-endorphin alone produced little effect in preventing CPK leakage into the blood.
Suitable active xcex2-endorphin fragments for use in the present invention are fragments at the C-terminal end of the xcex2-endorphin molecule, such as xcex2-endorphin (30-31; glycylglutamic acid), (29-31) and (28-31). In xcex2-endorphin, the amino acids 28, 29, 30 and 31 are lysine, lysine, glycine and glutamic acid (human) or glutamine (most other mammals), respectively.
As far as the analogues of the above active xcex2-endorphin fragments are concerned, stabilised analogues thereof are preferred wherein one or more of the following substitutions may be made:
Additionally, the hydrogen terminating the N-terminal end (preferably the xcex2-endorphin 28-amino acid-N-terminal) of the xcex2-endorphin fragment may be substituted by Ac, xcex2-Ala, HOOC(CH2)2COxe2x80x94, Tyr, benzylcarbonyl (C6H5CH2COxe2x80x94), malonyl or R (wherein Ac is an acyl group, for example acetyl, and R is a fatty acyl group.
Of such analogues, those where amino acid 30 (Gly) is replaced by Sar, those where amino acid 29 (Lys) is replaced by D-Lys, and those where the N-terminal (28) end of the fragment is Ac-Lys, are preferred. Particularly preferred is the stabilised analogue in which all three substitutions have been made, i.e. Ac-Lys-D-Lys-Sar-Glu, most preferably CH3CO-Lys-D-Lys-Sar-Glu.
Furthermore, the above C-terminal peptides (i.e. the above-mentioned xcex2-endorphin fragments are small molecules compared to xcex2-endorphin and analogues of these fragments can be made which are stable to proteolytic digestion and therefore have a relatively long half-life in the blood enabling their actions to be long lasting. Their resistance to proteolytic digestion may also make them effective via oral administration. In addition, the C-terminal peptides do not contain the opioid amino acid sequence and therefore are not likely to have the side effects of xcex2-endorphin which are due to its opioid actions.
Thus, it is considered that the active xcex2-endorphin fragments and analogues thereof may be administered intravenously, subcutaneously or intramuscularly, although stabilised analogues, such as Ac-Lys-D-Lys-Sar-Glu, may possibly be administered orally.
In one series of experiments, the C-terminal peptide, glycylglutamine, was injected intravenously into the blood of dystrophic mice 4 to 5 times a week in an amount of approximately 1.5 microgram/g body weight each time over a seven week period. Control mice were injected with the same volume of saline at the same times. There were six mice in each group.
The following measurements were made:
1. Change in body weight (the dystrophic mice would usually not gain significant weight over seven weeks and might even lose weight-depending on their age).
2. Weight of the extensor digitorum longus (EDL) muscle.
3. Level of CPK in the blood (a diagnostic test for muscular dystrophy is to measure the level of CPK in the blood. In muscle diseases, the blood levels of enzymes such as CPK are elevated and in muscular dystrophy the levels can be very high).
4. CPK enzyme levels in the muscle.
The following results were obtained:
1. The mice treated with glycylglutamine showed a statistically significant increase in body weight (of approximately 12%), whilst the saline controls showed a slight decrease in body weight (0.04% which is non-significant).
2. There were slight increases in EDL muscle weight (approximately 5%) which were not statistically significant.
3. The CPK level in the blood was reduced by 25%, indicating an improvement in the health of the muscle (i.e. less damage).
4. The enzyme concentration in the EDL muscle was increased by 55% (when calculated on a wet weight basis).
The above findings, particularly in result 4 above, indicate that chronic injections of this peptide could be therapeutic in muscular dystrophy. In Duchenne muscular dystrophyxe2x80x94the most common muscular dystrophy in humansxe2x80x94it appears to be the fast muscle fibres which are most involved in the disease process. The muscle examined (the EDL) is a fast muscle which contains a high proportion of fast fibres.
The effects of different xcex2-endorphin-derived peptides on the contractile responses in mouse diaphragm muscle have been further examined in order to see which regions of the parent molecule might be important for its effect.